A new procedure for isolating fibronectin matrix from chick embryo fibroblasts has been developed. Compared with the matrix isolated by previous procedures, the matrix prepared by the new procedures does not have detectable actin and myosin contamination. Coomassie blue staining of SDS polyacrylamide gel analysis of purified matrix revealed essentially only one protein component, fibronectin. However, by metabolic labeling with 32SO4 and 3H-glucosamine, there is a significant amount of glycosaminoglycans present in the isolated matrix. The major species of glycosaminoglycan are heparin sulfate and chondroitin sulfate. The isolated matrix has several unusual features: (1)\in contrast to previous report on matrix isolated from human fibroblasts, this matrix is resistant to 6 M guanidine hydrochloride as assessed by time-lapse video recording,immunofluorescent staining with fibronectin antibodies, FITC-gelatin binding, sedimentation in cesium chloride gradient, and electron microscopy; (2)\in contrast to the expectation, heparinase,chondroitinase AC, and chondroitinase ABC do not affect the fibrillar state of fibronectin matrix,nor do these enzymes liberate more than 10% of total radioactive sulfate and glucosamine present in the isolated matrix; (3)\the matrix is resistant to collagenase, 1% SDS, 8 M urea, and thrombin; and (4)\the fibrillar nature of this matrix is extremely sensitive to thiol-reducing agents. It appears that: (1)\fibronectin matrix from chick embryo fibroblasts is highly stabilized by disulfide linkages and (2)\glycosaminoglycans associated with fibronectin matrix may be protected from enzyme digestion by hitherto unknown mechanisms. The isolated fibronectin matrix has the following promoting effects: elongation and alignment of transformed fibroblasts, contact guidance, myoblast fusion, formation of very large cell aggregates (2cm in diameter), and restriction of carcinoma cell migration. (A)